Natural polyamines such as putrescine (1,4-butane-diamine), spermidine (N-3[aminopropyl]-1,4-diaminobutane) and spermine (N,N′-bis-[3-aminopropyl]-1,4-butanediamine) play essential roles in the control of macromolecular synthesis and growth processes in eukaryotic cells. Cells maintain appropriate polyamine concentrations principally by de novo synthesis from amino acids wherein ornithine decarboxylase catalyzes conversion of ornithine to putrescine, which is then converted to spermidine and spermine. Most tissues also possess a specific plasma membrane transport system allowing for utilization of plasma sources of polyamines.
Inhibitors of polyamine biosynthesis such as α-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase, cause an extensive depletion of polyamines followed by growth arrest in virtually all known mammalian cell types in vitro. Since tissues such as tumor cells and other transformed or rapidly proliferating cells exhibit a tissues such as tumor cells and other transformed or rapidly proliferating cells exhibit a high demand for polyamines, these properties have encouraged an extensive assessment of such inhibitors for the treatment of proliferative diseases, including several types of tumors, in experimental models and in clinical trials. Unfortunately, the antitumor efficacy of such inhibitors in vivo has been disappointing. The failure of DFMO to halt tumor growth in animal models has been clearly correlated with the elevated polyamine transport activity found in transformed cells. Indeed, decontamination of the gastrointestinal tract, which is the main vector of circulating polyamines through bacterial microflora activity, along with a polyamine-free diet, markedly potentiate the in vivo efficacy of DFMO against tumor progression. Moreover, mutant mouse leukemia cells deficient in polyamine transport are much more susceptible than the parental strain to growth inhibition by DFMO treatment in host animals. Besides, growth inhibition associated with DFMO-induced polyamine depletion in ZR-75-1 human breast cancer cells can be completely reversed by concentrations of spermidine as low as 300 nM, i.e., such as those found in human plasma (Moulinoux, J. -P., Quemener, V., and Khan, N. A. 1991. Cell. Mol. Biol. 37: 773-783; Scalabrino, G. and Ferioli, M. E. 1981. Adv. Cancer Res. 36: 1-102; Bachrach, U., 1989, in The Physiology of Polyamines (Bachrach, U. and Heimer, Y. M., eds.) Vol. II, pp. 235-249, 2 vols, CRC Press, Boca Raton, Fla.). The striking efficiency of the transport system to salvage exogenous polyamines in DFMO-treated cells owes to its upregulation consecutive to polyamine depletion (Seiler, N. and Dezeure, F. 1980, Int. J. Biochem. 22: 211-218; Byers, T. L. and Pegg, A. E. 1990, J. Cell Physiol. 143: 460-467; Lessard, M., Zhao, C., Singh, S. M. and Poulin, R. 1995, J. Biol. Chem. 270: 1685-1694; Kakinuma, Y., Hoshino, K., and Igarashi, K. 1988, Eur. J. Biochem. 176: 409-414). These data reinforce the view that cellular import of exogenous polyamines is the main factor limiting the efficacy of DFMO and other polyamine biosynthesis inhibitors as antitumor agents in vivo (Sarhan, S. Knödgen, B., and Seiler, N, 1989, Anticancer Res. 9: 215-224; Hessels, J., Kingma, A. W., Ferwerda, H., Keij, J., Van der Berg, G. A., and Muskiet, F. A. J. 1989, Int. J. Cancer 43: 115-1166; Ask, A., Persson, L. and Heby, O. 1992, Cancer Lett. 66: 29-34; Seiler, N., Sarhan, S., Grauffel, C., Jones, R., Knödgen, B. and Moulinoux, J. -P. 1990, Cancer Res. 50: 5077-5083; Persson, L., Holm, I., Ask, A. and Heby, O. 1988, Cancer Res. 48: 4807-4811).
Depletion of intracellular polyamines in tumor cells is thus a well-known strategy in anticancer therapies. However, it is now of common knowledge that depleting intracellular polyamines generally enhances polyamine uptake. To date, molecular information on the carrier molecules of the mammalian polyamine transport system is still unavailable. A few attempts have been made previously to design specific inhibitors of polyamine transport. Based on the finding that paraquat (4,4′-bipyridine) is a substrate of the putrescine transport system (Smith, L. L. and Wyatt, I. 1981, Biochem. Pharmacol. 20, 1053-10581; Rannels, D. E., Pegg, A. E., Clark, R. S. and Addison, J. L. 1985, Am. J. Physiol. 249, E506-E513), a series of polypyridinium salts, including compounds with a low Ki against putrescine uptake and low acute toxicity for mammalian cells have been synthesized (Minchin, R. F., Martin, R. L., Summers, L. A. and Ilett, K. F. 1989, Biochem. J. 262, 391-395). However, it is unclear whether such compounds can efficiently inhibit polyamine transport or are accumulated intracellularly. A number of polyamine analogs are effective competitors of polyamine uptake while being themselves substrates for transport (Seiler, N. and Dezeure, F., 1990, Int. J. Biochem. Cell. Biol. 27: 425-442; Bergeron, R. J., and Seligsohn, H. W. (1986) Bioinorg. Chem. 14: 345-355; Porter, C. W., Bergeron, R. J. and Stolowich, N. J. 1982, Cancer Res. 42: 4072-4078; Porter, C. W., Basu, H. S., Feuerstein, B. G., Deen, D. F., Lubich, W. P., Bergeron, R. J., Samejima, K., and Marton, L. J. 1989, Cancer Res. 49: 5591-5597; Pegg, A. E., Wechter, R., Pakala, R., and Bergeron, R. J. 1989, J. Biol. Chem. 264: 11744-11749; Pegg, A. E., Nagarajan, S., Naficy, S. and Ganem, B. 1991, Biochem. J. 274: 176-171; Porter, C. W., Ganis, B., Libby, P. R. and Bergeron, R. J. 1991, Cancer Res. 51: 3715-3720).
More recently, a high-molecular weight (Mr=25 kD) spermine polymer has been described by Aziz et al. in U.S. Pat. No. 5,456,908, as a competitive inhibitor of polyamine transport, with a Ki in the 10−6M range. In this patent document are disclosed two novel classes of polyamine transport inhibitors of high molecular weight, namely polymeric conjugates of normally transported substances (TS) of the structure (TS)m or conjugates of a polyamine and a protein or polypeptide (P) linked by known coupling agents and represented by (TS)-(P), wherein the repeating units of the polymer comprise the targeted polyamine. It is predictable that the inhibitors of Aziz et al. would be difficult to eliminate in vivo due to their high molecular weight and the high positive charge of the polymers, notwithstanding the risk of immunogenicity inherent to such high molecular weight inhibitors. The length of the polymers of Aziz et al. as well as their charge would cause their adsorption to the cellular surface, which bears negative charges due to the presence of glycoproteins, e.g. sialic acid. Poly-L-lysine, a commercially used compound analogous to high molecular weight polymers of polyamines by its positive charges, is known to promote a strong electrostatic interaction between the cell and its substrate, as in the induction of positive charges of gamma irradiation of synthetic polymers used to produce dishes for tissue culture. The polyamine transport inhibitors of Aziz et al. present the additional drawback of being highly cytotoxic. It is noteworthy that their spermine polymer is effective in decreasing contents of polyamines in cells even when not used in combination with DFMO and at concentrations much higher than those required to block polyamine uptake, which indicates inherent high toxicity of the compound toward the cell by a mechanism independent of polyamine transport per se. The cytotoxicity of the spermidine polymer of Aziz et al. is most probably explained by a non-specific effect on cellular physiology such as the cellular membrane. Although the authors pretend to demonstrate the specific action of the polymers with the fact that exogenous spermidine reverses the induced cytotoxicity, it is highly likely that competition between spermidine and the polymers modifies the electrostatic interaction with the negatively-charged sites on the cellular membrane is responsible for the effect. The results obtained by Aziz et al. indicate that at least part of the effect observed with high molecular weight polymers is non-specific (Aziz, S. M., Tofiq, S. F., Gosland, M. P., Olson, J. W. and Gillespie, M. N. 1995, J. Pharmacol. Exp. Ther. 274, 181-196). The usefulness of this spermine polymer for specificity blocking polyamine accumulation is therefore uncertain in view of its marked cytotoxicity.
Cysteamine and aliphatic monoamines of similar chain length such a n-butylamine and n-pentylamine have a low but significant ability to antagonize putrescine uptake (Gordonsmith, R. H., Brooke-Taylor, S., Smith, L. L. and Cohen, G. M. 1983, Biochem. Pharmacol. 32, 431-437), although the mode of inhibition of these compounds has not been reported. The only polyamine-like structure known to interact non-competitively with the polyamine transport system is pentamidine, an aromatic diamidine (Jones, H. E., Blundell, G. K., Wyatt, I., John, R. A., Farr, S. J. and Richards, R. J. 1992, Biochem. Pharmacol. 43, 431-437), but the structural basis of its inhibitory activity is not yet clear.
It follows that there still exists a need for effective polyamine transport inhibitors which, while inhibiting the transport of polyamines, will not be internalized by the transport system and will not be toxic to the cell. The availability of low molecular weight inhibitors of polyamine transport would provide for the possibility of better renal elimination, as well as lower risks of being immunogenic. The availability of high-affinity, specific, but impermeant antagonists of polyamine transport would also allow to evaluate the antitumor efficacy of polyamine depletion strategies in vivo with minimal systemic cytotoxic effects.
There is much preclinical evidence supporting the hypothesis that the efficacy of the suicide inhibitor of ornithine decarboxylase, D,L-α-difluoromethylornithine (DFMO=Eflornithine) as a chemotherapeutic agent is limited by the enhanced ability of tumor cells to transport polyamines from plasma sources. Plasma polyamines are partly derived from various plasma sources (7, 12, 18, 58-60, 62, 70) and from the activity of the gastrointestinal microflora, which produces and excretes very high amounts of putrescine and cadaverine (1, 17, 45, 50, 62, 70), which can enter the general circulation through the enterohepatic pathway (6, 45). Other systemic contributions can also be attributed to polyamine excretion by peripheral tissues, including dying tumor cells (32, 35, 41, 42, 63, 64, 67, 79, 80). The enhanced uptake of polyamines by tumor cells results both from the increased polyamine transport activity that accompanies the malignant phenotype (11, 43, 51, 68, 69), and from the effect of DFMO itself, which causes a compensatory upregulation of polyamine uptake across the plasma membrane (9, 10, 14-16, 22, 25, 29, 31, 38, 39, 43, 47, 48, 50, 57, 61). One possible strategy that could be used to overcome this phenomenon would be to administer a pure antagonist of polyamine transport, i.e. a drug which binds with high affinity to the polyamine transporter, but which cannot be transported by this membrane protein. While a need continues to exist for such compound, no such compound is yet available.